Epidemiology and Performances of Typhidot Immunoassay and Widal Slide Agglutination in the Diagnosis of Typhoid Fever in Febrile Patients in Bafoussam City, Cameroon: A Cross-Sectional Comparative Study

Background Enteric fever is a great public health problem associated with significant illness and death in many endemic countries, and its clinical diagnosis is still daunting. The aim of this study was to determine the prevalence and risk factors of S. Typhi among febrile patients in Bafoussam and to evaluate the diagnostic performances of Widal and Typhidot tests. Methods This was a cross-sectional study among 336 participants visiting three hospitals in Bafoussam from August 1, 2021, to November 31, 2021. Widal test, Typhidot assay, and stool culture were used to screen for salmonellosis with the help of a structured questionnaire. Results The prevalence of S. Typhi and S. Paratyphi was found to be 62.85% and 37.14%, respectively. The overall prevalence of typhoid fever using stool culture was 20.86%. The significant risk factors associated with enteric fever were lack or insufficient knowledge of typhoid fever, poor hand hygiene, and anorexia. Typhidot immunoassay was more sensitive (100%) and specific (82.3%) than the Widal test. Both were analytically inferior to stool culture. Conclusions High prevalence of typhoid fever (20.86%) was observed which was largely associated with lack or insufficient knowledge of typhoid fever, poor hygiene measure, and anorexia as risk factors. The performances of the Widal and Typhidot test against a stool culture were inferior but with Typhidot better than the Widal slide agglutination.


Introduction
Typhoid or enteric fever is a systemic, bacterial, potentially severe infection linked to poor hygiene practices.It is caused by Salmonella Typhi, a Gram-negative bacterium.It has become uncommon in developed countries due to progress in hygiene and improved water supply conditions [1,2].However, it is one of the main causes of hospital admissions in developing countries [3,4].Te disease is transmitted via the fecal-oral route from Salmonella contaminated food or dirty water [5].Te main and common symptoms of the disease include malaise, fever, vomiting, constipation, splenomegaly, and hepatomegaly [6].Te disease can lead to serious complications such as internal bleeding and intestinal perforation [7].However, despite the decline in morbidity and mortality from typhoid fever in developed countries, it remains one of the main major public health problems in developing countries, particularly in Central Africa where it is endemic [2].
Te real combat in the treatment of typhoid fever lies in its misdiagnosis in the clinical setting due to overlapping symptoms with other common infections such as malaria, dengue, and viral diseases [8] Accurate and rapid diagnosis of typhoid fever depends on isolating the pathogen and identifying potential carriers of the infection [9].Detection of S. Typhi can be done from many body fuids and biomaterials such as blood, stool, urine, bone marrow, rose spot extracts, duodenum, and aspirates [10].Blood, stool, and urine cultures are the best diagnostic methods, but they are very costly techniques and require equipment and qualifed personnel who are often lacking in many clinical training institutions in Cameroon.Te diferent methods employed to diagnose typhoid fever are clinical signs and symptoms, the use of serological markers (Widal test), bacterial culture (stool culture and blood culture), antibody detection (Typhidot), and molecular methods (amplifcation of DNA by PCR) [11].Blood culture and stool culture are the two most specifc tests in the diagnosis of typhoid fever [12], the gold standard for diagnosing typhoid fever is blood culture, but it is expensive for patients, time-consuming, and in remote rural settings, culture facilities may be unavailable [13].Stool culture could also be used where blood culture is inaccessible because of a strong agreement between blood and stool culture for diagnosing typhoid fever [14].Widal slide agglutination test is very simple and afordable to perform and requires minimal training and equipment and has been used in the diagnosis of typhoid fever for a long time in Cameroon.It remains a very common and good serological test with a moderate specifcity, but its sensitivity in the diagnosis of typhoid fever is hard to interpret for several reasons: delayed time between infection and antibody production, cross-reactivities, and the long stay of target serum antibodies after treatment with lack of active disease [15].Typhidot immunoassay is a rapid serological assay for the diagnosis of enteric fever and can be used as an alternative to Widal test due to its better sensitivity and specifcity [16].However, Typhidot is not suitable to replace blood culture [8].Can Typhidot be better than stool culture is the question we ask ourselves?Stool culture is still dominantly used as a confrmatory test for typhoid fever in most sub-Saharan clinical facilities owing to its low cost relative to hemoculture.Tis study was carried out to determine the prevalence and risk factors of S. Typhi among patients in Bafoussam and to evaluate the diagnostic performances of Widal test and Typhidot immunoassay visà-vis stool culture among febrile patients in the city of Bafoussam.

Study Area and Period.
Te city of Bafoussam is located on the Bamileke plateau at 1420 m altitude and 5 °28′ north latitude.Te Department of Mif is one of the six departments of the West Region of Cameroon with Bafoussam as its capital.Tis department is divided into three districts as follows: Bafoussam 1, Bafoussam 2, and Bafoussam 3 (Figure 1).Its population constitutes 465,000 inhabitants with an area of 402 km 2 with a long rainy season and a short dry season.Precipitation there varies around 1717.7 mm, and temperatures vary around 13.6 °C to 25.35 °C.Te population's livelihood is mainly on agriculture, livestock, and trade.Te main agricultural products are corn, beans, potatoes, carrots, cabbage, banana, and plantains.Te main animals raised are chickens and pigs.
Tis study took place during the period from August 1, 2021, to November 31, 2021.

Study Design and Population.
A cross-sectional study was conducted in three hospitals in the city of Bafoussam (Bingo Cameroon Baptist Hospital of Bamendi; Mif District Hospital and Catholic Health Center of Lafee-Baleng) on patients with symptoms of gastroenteritis or typhoid fever.All eligible participants (temperature >37.5 °C at inclusion) who gave their written informed consent were recruited and attributed a unique study code.Tis code was used for all samples collected and diferent for each patient.A questionnaire containing demographic, clinical, and lifestyle information was administered to the patient.Tree hundred and thirty-six patients were recruited based on the criteria listed in Table 1.

Blood Collection.
Using a sterile syringe and needle, 3 mL of venous blood was drawn from each patient's forearm into a dry tube prelabeled with an anonymized patient code.Te analysis of the blood samples was done by a qualifed laboratory technician.

Test Procedure for the Typhidot Immunoassay Test.
Typhidot test (Medsource ozone bio medicals, Faridabad, India) is an immunochromatographic assay designed for simultaneous detection and diferentiation of specifc IgM and IgG antibodies against S. Typhi in human serum.Te diagnostic test cassette consists of two components: an IgG component and an IgM component.Te (G) line region is precoated with reagents for the detection of anti-S.Typhi (IgG) and the (M) line region is precoated with monoclonal antihuman IgM for detection of anti-Salmonella Typhi (IgM).Te (C) line corresponds to the negative control.During testing, serum is dispensed into the sample well and antibodies in serum bind to typhoid antigen conjugates impregnated in the reagent area if the serum contains antityphoid antibodies.
Using a sterile syringe and needle, 3 mL of venous blood was collected from each patient's forearm into a dry tube prelabeled with an anonymized patient code.Te blood specimens were then spun at 3000 rpm for 5 min using a laboratory centrifuge.Using the provided pipette, one drop of serum was added to the test well on the test cassette followed by one drop of bufer.Te setup was allowed for 15 minutes for migration across the membrane and color development in the result windows.A positive IgM was interpreted clinically as acute typhoidal disease, while IgM and IgG positive were taken as acute typhoidal fever disease in the middle stage of infection.IgG positive band indicated a chronic carrier or previous infection or reinfection.A colored control line (C) must always appear in case of a negative or a positive result.Its absence indicates invalid test results.Table 1: Summary of study participant recruitment criteria.
Inclusion criteria a (N � 410) Noninclusion criteria (49) b Exclusion criteria c (n � 25) (i) Patients with fever, temperature ≥37.50 C (i) Subjects with hemolyzed blood (ii) Subjects of at least one year old, regardless of sex or ethnicity, who have given a favorable opinion to participate freely in the study, or parental consents (i) Patients on antibiotic treatment (ii) Subjects who refused to sign the informed consent form (ii) Patients who did not provide all the required information (iii) Febrile subjects with malaria and respiratory infections a Inclusion criteria are characteristics that the prospective subjects must have if they are to be included in the study.b Noninclusion criteria are characteristics that subjects must not have if they are to be included in the study.c Exclusion criteria are features that exclude subjects from the study in progress.
laboratory.Approximately 4 g of fresh stool were collected from each patient suspected to have Salmonella infections for the coproculture test after a brief training on how to collect the sample using a sterile wide-mouthed and aseptic transparent container.Materials were provided to each of the participants for stool collection.After collection, the stool was emulsifed in 3 ml normal saline and 1 ml of this inoculum was diluted with 9 ml of freshly prepared Selenite F Broth (Oxoid CM0395B and LP0121A) in prelabeled tubes and incubated at 37 °C for 24 hours aerobically in an incubator for bacterial growth.A loop full of inoculum from 3 ml normal saline was inoculated by the streaking method onto Salmonella Shigella selective agar (SSA) (Neogen, Marshfeld, United States) and MacConkey agar plates (McConkey and Co, Puyallup St, United States).Incubation of all culture plates were carried out at 37 °C for 24 hours.Isolation of S. typhi in stool culture indicated an infection.In the absence of growth, the culture was considered negative and subcultures were repeated for one week.Isolates were confrmed to be S. typhi and S. Paratyphi by API20E gallery (bioMerieux, Marcy-l' Étoile France) and by agglutination with Salmonella agglutinating sera [17].Te qualifed laboratory personnel who performed coprocultures were blinded to the procedure and results of the Widal, Typhidot, and stool culture tests.

Quality Control.
All the instruments used for sample processing were checked every morning for proper functioning, and standard procedures were followed during processing of each sample.

Ethical Approval. Cameroon National Committee for
Ethics in Human Health (Ref no 2022/08/128/CE/CNERSH/ SP) approved the experimental procedures and protocols used in this study.Te concept of the study was explained to the participants, and written informed consent was obtained from all participants before enrolment.A designed questionnaire was administered to each participant to collect clinical symptoms, hygienic, behavioral, and demographic information.

Statistical Analyses
Te sample size was calculated based on the nomogram published by Carley et al. (2003).Based on this nomogram, we calculated the sample size by using the following criteria: an estimated prevalence of typhoid fever of 31%, a sensitivity plot at alpha � 0.05, a confdence limit of 95%, and a type 1 error of 0.05.We obtained for this study an estimated sample of 336 patients.Collected data were entered into Excel and exported to SPSS (Statistical Package for Social Science) version 24.0 for further analysis.Te chi-squared (X 2 ) test was used to check the statistical signifcance, and signifcance was tested at p ≤ 0.05.Te sensitivity, specifcity, and predictive values were calculated based on standard formulae, while the area under the receiver operating curve was used to estimate the diagnostic accuracy of the Widal and Typhidot tests.Kappa test was used to assess the agreement between the tests.Te test accuracy, sensitivity, specifcity, positive predictive value, and negative predictive value were compared to Typhidot immunoassay and Widal slide agglutination test against stool culture.Logistic regression analysis was used to determine associations of sociodemographic, hygienic, behavioral, and clinical characteristics with typhoid fever.

Results
Of the 410 patients admitted and screened for clinical features of febrile illness or history of fever, 74 were excluded from the study, viz., 10 were nonfebrile illness patients, 25 were patients on antibiotic treatment, 11 were patients who refused to sign the informed consent form, and 28 were patients who did not provide all the required information.Te stool cultures were collected from 336 patients who met the inclusion criteria (Figure 2).Out of the 336 patients included, 70 were positive for Salmonella enterica in whom 44 for Salmonella typhi and 24 for Salmonella typhi infection.

Distribution of the Prevalence of Typhoid Fever in the Study
Population.Table 3 presents the prevalence of typhoid fever according to sociodemographic characteristics and reveals a signifcant diference (p � 0.01) between occupation and Salmonella typhi and S. Paratyphi infection.
Indeed, there was a higher prevalence of S. typhi and S. Paratyphi among private sector workers.Characteristics such as sex, age, and level of education were not signifcantly related to S. typhi and S. Paratyphoid infections.Tis table also shows the prevalence of typhoid fever according to lifestyle.We noted a signifcant diference between the level of knowledge of typhoid fever and infection with S.     Values in brackets are in percentage (%).In column three, the denominator is 44.In column four, the denominator is 26.Numbers in bold represent the P value of each sociodemographic characteristic which is signifcantly diferent compared to the normal P value (0.05). 8 Canadian Journal of Infectious Diseases and Medical Microbiology knowledge on environment, routes of transmission, hygiene measures taken against typhoid, type of drinking water used, and measures taken to make water drinkable were not linked to the infections of S. typhi and S. Paratyphi.
Regarding the signs and symptoms, a signifcant difference was observed between vomiting, fever, constipation, anorexia, and infection with S. typhi and S. Paratyphi with a p value of 0.01, 0.03, 0.008, and 0.02, respectively (Table 4).Parameters such as headache, body aches, chills, diarrhea, asthenia, type of stool, and abdominal pain did not show a signifcant diference with S. typhi and S. Paratyphi infections (Table 3).

Salmonella Serotypes. Figure 3 illustrates the diferent
Salmonella serotypes isolated from febrile patients.API 20E Gallery used identifed fve main types of Salmonella strains including Salmonella typhi, Salmonella Paratyphi A, Salmonella Paratyphi B, Salmonella Paratyphi C, and Salmonella typhimirium.Te S. typhi strain was the most represented (71.42%) followed by the S. Paratyphi A strain (12.85%), S. Paratyphi C (4.28%), and S. typhimirium (4.28%) strains which were the least represented.

Associated Risk Factors of Typhoid Fever.
Sociodemographic parameters such as gender, age groups, level of education, marital status, occupation, and residence were used to determine the risk factors.A logistic regression analysis was performed to investigate the association between these factors and typhoid fever.None of the assessed sociodemographic parameters were found to be signifcantly associated with typhoid fever (Table 4).

Lifestyle and Clinical Symptoms
4.5.1.Lifestyle.Knowledge of typhoid fever was signifcantly associated with typhoid fever (p � 0.043).Indeed, the logistic regression showed that the absence or insufcient knowledge of typhoid fever in patients exposed them to typhoid fever (50%) compared to those with knowledge (20.12%) (Table 5).Lack of knowledge of typhoid fever strongly predicted exposure to typhoid fever (OR � 5.243, 95% CI: 1.052-26.119).Regarding hygiene measures against typhoid fever, multivariate logistic regression showed that patients who did not wash hands after bowel movements were positively associated with typhoid fever (3.740, 95% CI: 1.515-9.234).
Headache, vomiting, muscular pain, fever, frisson, diarrhea, constipation, asthenia, type of stool, and abdominal pain were not found to be associated with typhoid fever (Table 7).

Comparison of Typhidot, Widal Slide Agglutination Test with Stool Culture in Diagnosis
We compared Widal and Typhidot in their ability to diagnose typhoid fever relative to stool culture.6).

Prevalence of Typhoid Fever Based on Tree Diagnostic
Methods in the Study Population.Table 9 summarizes the diferences in prevalence estimates by the three tests.Out of the 336 participants in this study, 159 were positive for typhoid fever with the Widal slide agglutination test with an antibody titer of 1 : 80 for both "O" and "H" antigens being taken as cutof point values indicative of a recent typhoid infection.Tis gives a prevalence of 47.32% and 26.78% (n � 90) who was negative for typhoid fever when stool culture was used as the reference diagnostic tool.With the Typhidot-IgM detection method, 117 were positive with a prevalence of 34.82% and 13.98% (n � 47) who were negative for typhoid fever when stool culture was used as the reference diagnostic tool.Te diference in estimating the prevalence of typhoid fever was signifcant (p < 0.0001) when comparing the four diagnostic methods.Also, the Widal test identifes 47.32% of febrile patients suspected of typhoid fever as positive, whereas only 26.86% of these patients were positive for the Typhidot immunoassay.Te low accuracy of the Widal agglutination test compared to Typhidot could be explained by exposure to crossreacting antigens in febrile infections other than Salmonella typhi.Typhidot test identifes 34.82% of febrile patients suspected of typhoid as positive, whereas only 13.96% of these patients were positive for the reference test.
Canadian Journal of Infectious Diseases and Medical Microbiology

Discussion
Typhoid fever continues to be a menacing cause of illness and death worldwide, especially in countries with insufcient resources such as Cameroon.Te clinical diagnosis of enteric fever is not easy to arrive at owing to presentation of signs and symptoms that may overlap with febrile illnesses in regions where typhoid is endemic [18].Tis work is the frst study done on the association of risk factors to typhoid fever in this part of Cameroon.
In this study, a total of 336 samples were taken from patients attending three main hospitals of the West region of Cameroon, with the mean age of 25.20 ± 16.77 years.Te prevalence of typhoid fever among febrile patients in the three diferent health centers of Bafoussam with stool culture was 20.86% indeed high.Tis fnding was similar to that obtained in Buea-Cameroon (21%) [19] but higher than studies conducted in Ethiopia (5%) [20] and 15.3% [21] and in Indonesia (15.5%) and Lalitpur 4.1% [18].Tis high prevalence of typhoid fever observed in this study can be attributed to the nonspecifc clinical symptoms that can overlap with febrile illnesses in regions where typhoid is endemic [22].Lack or insufcient knowledge of typhoid fever and hygiene measures such as not washing hands after bowel movements and anorexia may explain this prevalence rate.Participants who lacked or had insufcient knowledge of typhoid fever were found to be signifcantly more infected by typhoid fever pathogens.Tis might be explained by their level of education where primary and secondary young students were more infected and had poor practices of the hygiene measures against typhoid.Tis suggests that vital information on the knowledge and prevention of the disease is needed in the health districts in Cameroon via periodic sensitization seminars.Talking of the hygiene measures against typhoid fever, a statistically signifcant association was observed between washing hands after bowel movements and typhoid or paratyphoid infection positivity.We noted that most participants who tested positive for typhoid failed to wash their hands regularly.Tus, lack of hand hygiene is a great risk factor to contracting and transmitting enteric infections.Tis notion is buttressed by an earlier report which demonstrated that those who prepared food and operated restaurants did not perform hand hygiene after toilet use and constituted a transmission risk factor to those who visited to eat, thus establishing a chain of infectivity for typhoid fever [23].Our fndings are also endorsed by the dramatic story of the typhoid fever carrier cook, Mary Malon, who inadvertently contaminated 7 out of 8 families during her professional cooking career [24].All these observations and reports suggest that hand washing with soap after bowel movements is very important to prevent typhoid fever [25].Analysis by symptoms and clinical fndings showed anorexia was associated to typhoid and contributed to the diagnosis in patients with typhoid fever.Tis result is in accordance with the study carried out by [26] in Douala-Cameroon.In column two, the denominator is 336.In column three, the denominator corresponds to the total of each variable in the lifestyle.Numbers in bold represent the P value of each lifestyle which is signifcantly diferent compared to the normal P value (0.05).Te diagnosis of typhoid has relied on the isolation of Salmonella species from blood, feces, urine, and bone marrow, with the isolation rate varying from 30% to 70%.
Isolation of the causative agent by culture has remained the gold standard for diagnosis of typhoid fever [8].However, it is well recognized that facilities for culture are not readily   [13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28].Low sensitivity for the Widal agglutination test can be related to the data collection time.Te Widal test correctly identifes many patients who are uninfected with S. typhi and S. Paratyphi (high specifcity), while only about 26.48% of uninfected patients will be wrongly classifed as having typhoid fever (false positives); meanwhile, they are actually uninfected.So, a negative Widal agglutination test result has a good predictive value for the absence of typhoid fever, but a positive result would have a low predictive value for the presence of disease [29].Also, the Widal test identifes 47.32% of febrile patients suspected of typhoid fever as positive, whereas only 26.86% of these patients were positive for the Typhidot immunoassay.Te low accuracy of the Widal agglutination test compared to Typhidot could be explained by exposure to crossreacting antigens in febrile infections other than Salmonella typhi [30].Te Typhidot test identifes 34.82% of febrile patients suspected of typhoid as positive, whereas only 13.96% of these patients were positive for the stool culture reference test.Tis observation could be due to early sampling before antibodies become detectable in blood as was reported in one study [13].Moreover, in this study, Typhidot-IgM and stool culture analyses agree more by 70.26% than with the Widal agglutination test.A recent report has shown that stool antigen RDT concurs by 86.7% with stool culture [21].Tis suggests that antibody and antigen detection by RDTs is credible and needs optimization of the technology to yield more sensitive results.Furthermore, stool culture, though with its own shortcomings, still stands as a better reference test in resourcedefcient and low-income countries where blood culture and PCR would be very expensive for the local population.
Our study had some limitations.Tis study regrets meeting up with the use of the reference standard (blood culture or PCR) owing to lack of funding or limited resources.Te low number of participants which could greatly afect the statistical power of the study was not attained.Responses provided in the questionnaire for age group below 12 years were given by parents and guardians who could introduce recall bias in the study.

Conclusion
Te overall prevalence of typhoid fever based on stool culture was 20.86%, and this was associated with lack or insufcient knowledge of typhoid fever, absence of hand washing after toilet use, and anorexia as clinical symptoms.Te performance of the Widal slide agglutination test and Typhidot immunoassay against a stool culture showed analytic inferiority.Typhidot immunoassay was more sensitive (100%) and specifc (82.3%) than the Widal test and is a better alternative in the diagnosis of typhoid fever.Tere was a substantial agreement between the Typhidot-IgM technique and stool culture.Stool culture remains a better assay than Typhidot and a better reference assay in resourcelimited settings.Tus, we recommend the use of Typhidot immunoassay in the detection of enteric infections in Cameroon's healthcare institutions and the great reduction of the use of Widal assay where appropriate.researchers who conceived the study, designed and collected data, conducted data analysis, and drafted the manuscript for publication.Aurelie Dahlia Yemeli Piankeu conducted data collection and technical works.Aurelie Dahlia Yemeli Piankeu, Emmanuel Boris Gomseu Djoumsie, Georges Ful Kuh, Siméon Pierre Chegaing Fodouop, Michel Noubom, and Donatien Gatsing contributed in designing the study and analysis and interpretation of data and reviewed the initial draft manuscript.Donatien Gatsing oversaw the whole project.All authors read and approved the fnal manuscript.

Figure 1 :
Figure 1: Sample collection location sites in West Region (turquoise circles represent study sites).

Figure 2 :
Figure 2: Flow diagram of febrile patients with positive stool culture in Bafoussam, Cameroon.

Table 2 :
Sociodemographic characteristics and frequency distribution of the study population.
*Te denominator used to calculate the frequency in column three is 336.Canadian Journal of Infectious Diseases and Medical Microbiology

Table 3 :
Prevalence of typhoid fever by sociodemographic characteristics, lifestyle, and signs and symptoms in the study population.

Table 4 :
Multivariate analysis of sociodemographic characteristics of study participants.
*Values in brackets are in percentage (%).In this table, the denominator for column three corresponds to the total of each variable.

Table 5 :
Multivariable analysis of life style of study participants.
*Values in brackets are in percentage (%).

Table 7 :
Distribution of clinical symptoms among study participants.Numbers in bold represent the P value of each clinical symptom which is signifcantly diferent compared to the normal P value (0.05).
*Values in brackets are in percentage (%), denominator column 2 : 336, and denominator column 3 corresponds to the total of each clinical symptoms.*

Table 8 :
[8]]dity of Widal and Typhidot immunoassays as diagnostic tools in comparison with stool culture for the diagnosis of typhoid fever.Canadian Journal of Infectious Diseases and Medical Microbiology available in low-income or resource-limited countries.Te Widal test, commonly employed in our setting, has been available for decades and can be performed using venous blood.Unfortunately, the assay has low sensitivity and specifcity especially in endemic zones and optimally requires comparison of samples drawn at the acute and convalescent stage of illness[27].In a developing country like Cameroon, the Widal test has been used extensively in the serodiagnosis of typhoid fever and stool culture as confrmatory test.Tough blood and bone marrow cultures and PCR are considered as gold standard or reference[8], we could not aford to incorporate this standard.Nonetheless, stool culture and blood culture have been reported to have a strong agreement[14].Our results of the Widal test showed that out of 336 samples, 159 were positive for S. typhi and S. Paratyphi with a prevalence of 47.32%.Tis prevalence was 34.82% (117 cases) with Typhidot.Tese results revealed disparities in the analytic performances of the Widal slide agglutination and the Typhidot technique.